1. Field of the Invention
This invention relates generally to a catalytic conformational sensor method and application of such method for detecting proteins and proteinaceous particles; and more particularly to detecting misfolded or disease-associated proteins and proteinaceous particles.
2. Related Art
The present invention detects misfolded or abnormal conformations of proteins or peptides such as those contributing to “folding diseases”. The “folding diseases” are characterized by proteins with destabilizing conformers which tend to aggregate and eventually form toxic plaques in brain and other tissue. See Bucciantini, M., et al. (2002) Inherent Toxicity of Aggregates Implies a Common Mechanism for Protein Misfolding Diseases. Nature 416:507-511.
These “folding diseases” can be hard to diagnose since the disease symptoms may be latent where the aggregates are slowly building up over time and go through stages of increased aggregation leading to fibril formation and eventual plaque deposition leading to impairment of cellular viability. Such misfolding of peptides and aggregate formation is believed to play a key role in Alzheimer's disease where beta-amyloid protein (or A beta, a 39-42 residue peptide) forms fibrillar deposits upon a conformer change; Huntington's disease where insoluble protein aggregates are formed by expansion of poly-glutamine tracts in the N-terminus of huntingtin (Htt), an antiapoptotic neuronal protein; and noninfectious cancers such as in cases where tumor-associated cell surface NADH oxidase (tNOX) has prion-like properties such as proteinaseR, ability to form amyloid filaments and the ability to convert the normal NOX protein into tNOX. See Kelker, et al. Biochemistry (2001) 40:7351-7354. for more information on tNOX.
The present invention, however, is not limited to the detection of proteins or peptides in folding-disease or infectious samples. It also includes detection of proteinaceous particles such as prions. Prions are small proteinaceous particles with no nucleic acids, thus are resistant to most nucleic-acid modifying procedures and proteases. The normal prion (PrP) protein is a cell-surface metalloglyroprotein that is mostly an alpha-helix and loop structure as shown in FIG. 8, and is usually expressed in the central nervous and lymph systems. It's proposed function is that of an antioxidant and cellular homeostasis.
The abnormal form of the PrP, however, is a conformer which is resistant to proteases and is predominantly beta-sheet in its secondary structure as shown in FIG. 9. It is believed that this conformational change in secondary structure is what leads to the aggregate and eventual neurotoxic plaque deposition in the prion-disease process.
The abnormal prion are infectious particles that play key roles in the transmission of several diseases such as Creutzfeldt-Jakob syndrome, chronic wasting disease (CWD), nvCJD, transmissible spongiform encephalopathy (TSE), Mad Cow disease (BSE) and scrapie a neurological disorder in sheep and goats1. 1 Clayton Thomas, Tabor's Cyclopedic Medical Dictionary (Phil F. A. Davis Company, 1989), at 1485.
Diseases caused by prions can be hard to diagnose since the disease may be latent where the infection is dormant, or may even be subclinical where abnormal prion is demonstrable but the disease remains an acute or chronic symptomless infection. Moreover, normal homologues of a prion-associated protein exist in the brains of uninfected organisms, further complicating detection.2 Prions associate with a protein referred to as PrP 27-30, a 28 kdalton hydrophobic glycoprotein, that polymerizes (aggregates) into rod-like filaments, plaques of which are found in infected brains. The normal protein homologue differs from prions in that it is readily degradable as opposed to prions which are highly resistant to proteases. Some theorists believe that prions may contain extremely small amounts of highly infectious nucleic acid, undetectable by conventional assay methods.3 As a result, many current techniques used to detect the presence of prion-related infections rely on the gross morphology changes in the brain and immunochemistry techniques that are generally applied only after symptoms have already manifest themselves. Many of the current detection methods rely on antibody-based assays or affinity chromatography using brain tissue from dead animals and in some cases capillary immunoelectrophoresis using blood samples. 2 Ivan Roitt, et al., Immunology (Mosby—Year Book Europe Limited, 1993), at 15.1.3 Benjamin Lewin, Genes IV (Oxford Univ. Press, New York, 1990), at 108.
The following is an evaluation of current detection methods.
                Brain Tissue Sampling. Cross-sections of brain can be used to examine and monitor gross morphology changes indicative of disease states such as the appearance of spongiform in the brain, in addition to immunohistochemistry techniques such as antibody-based assays or affinity chromatography which can detect disease-specific prion deposits. These techniques are used for a conclusive bovine spongiform encephalopathy (BSE) diagnosis after slaughter of animals displaying clinical symptoms. Drawbacks of tissue sampling include belated detection that is possible only after symptoms appear, necessary slaughter of affected animals, and results that takes days to weeks to complete.        Prionic-Check also requires liquified-brain tissue for use with a novel antibody under the Western Blot technique. This test is as reliable as the immunochemistry technique and is more rapid, yielding results in six to seven hours, but shares the drawbacks of the six-month lag time between PrPs accumulation (responsible for the gross morphology changes) in the brain and the display of clinical symptoms, along with the need for slaughter of the animal to obtain a sample.        Tonsillar Biopsy Sampling. Though quite accurate, it requires surgical intervention and the requisite days to weeks to obtain results.        Body Fluids: Blood and Cerebrospinal Sampling. As in the above detection methods, results are not immediate        Electrospray ionization mass spectrometry (ESI-MS), nuclear magnetic resonance NMR, circular dichroism (CD) and other non-amplified structural techniques. All of these techniques require a large amount of infectious sample, and have the disadvantage of requiring off-site testing or a large financial investment in equipment.        
The following is a survey of currently approved and certified European Union (EU) prion-detection tests.                Prionics—in Switzerland. The test involves Western blot of monoclonal antibodies (MABs) to detect PrP in brain tissue from dead animals in seven to eight hours.        Enfer Scientific—in Ireland. The test involves ELISA-based testing on spinal cord tissue from dead animals in under four hours.        CEA—in France. The test involves a sandwich immunoassay using two monoclonals on brain tissue collected after death in under twenty-four hours.        
The EU Commission's evaluation protocol has sensitivity, specificity and detection limits and titre. The sensitivity of a test is the proportion of infected reference animals that test positive in the assay. It previously used 300 samples from individual animals to assess this element. The specificity of a test is the proportion of uninfected reference animals that test negative in the assay. Previously used 1,000 samples from individual animals for this purpose. In order to test detection limits, various dilutions ranging from 100 to 10−5 of positive brain homogenate were used. A table showing an evaluation of EU test results is shown in FIG. 12. Even with high degrees of sensitivity and specificity, however, the fact remains that these tests must be performed post-mortem and require working with large amounts of highly infectious biohazard materials.
The Center for Disease Control (CDC) classifies prions as Risk Group 2 agents requiring Biosafety Level 2 (BSL2) containment. As a result many of the above operations are carried out under BSL2 physical containment with elevated safety practices more typical of a BSL3 lab. Prions can be inactivated by fresh household bleach, 1 molar NaOH, 4 molar guanidine reagents, or phenol followed by 4.5 hours of autoclaving at 132° C. Procedures involving brain tissue from human patients with neurological degenerative disorders pose special challenges and should be handled with the same precautions as HIV+ human tissue. Thus, working with large amounts of such biohazardous materials can be an obstacle to quick and simple testing of mass quantities or assembly-line samples as well as cumbersome even for small applications.
In addition to working with relatively large amounts of biohazardous materials and taking several hours to weeks for detection, many of the prior art methods have the added difficulty that they are performed post mortem.
As can now be seen, the related art remains subject to significant problems, and the efforts outlined above—although praiseworthy—have left room for considerable refinement. The present invention introduces such refinement.